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. 2017 Dec 19;47(6):1083–1099.e6. doi: 10.1016/j.immuni.2017.11.016

Figure 7.

Figure 7

Restoration of Tumor Cell TTP Expression Enhances Anti-tumor Immunity

(A) Western blotting analysis of CT26 Myc-TTP tet-ON cells expressing either empty vector or mouse Cd274 cDNA lacking the 3′ UTR (PD-L1 Δ3′ UTR), 24 hr after treatment (Dox., 0.1 μg/mL or 1 μg/mL). Arrow indicates Myc-TTP. Data are representative of two independent experiments.

(B) Representative flow cytometry histograms of PD-L1 surface protein expression in CT26 stable cells lines in (A), 72 hr after treatment (Dox., 1 μg/mL). Data are representative of three independent experiments.

(C) Tumor growth curves for CT26-derived cell lines subcutaneously transplanted into BALB/c mice (n = 8 per group).

(D) Tumor growth curves for MC38-derived cell lines subcutaneously transplanted into C57BL/6 mice (n = 6 per group). X denotes the loss of a doxycycline-treated mouse.

(E) Tumor growth curves for CT26-derived cell lines subcutaneously transplanted into nu/nu mice (n = 6 per group).

(F) Tumor growth curves for CT26-derived cell lines subcutaneously transplanted into BALB/c mice (n = 4–5 per group).

For (C)–(F), data represent the mean ± SEM from individual experiments. ∗∗p < 0.01, ∗∗∗∗p < 0.0001, n.s., not significant; two-way ANOVA.

(G) Histological analysis of subcutaneous tumors at the end-point from the experiment described in (C), with quantification of CD3+ cells in 5 fields of view per mouse with 5–6 mice per group. Mean ± SEM. ∗∗p < 0.01; unpaired, two-tailed Student’s t test.

(H) Quantification of CD8+/Treg cell ratios and CD8+ IFN-γ+ cells from flow cytometry analysis of tumors after 18–20 days of growth. Each data point represents data from an individual mouse; mean ± SEM. ∗p < 0.05; unpaired, two-tailed Student’s t test. Data are pooled from two independent experiments.

(I) Proposed molecular model. Signaling nodes that influence anti-tumor immunity and are amenable to inhibition with drugs used in this study are highlighted. S52 and S178 represent MK2 target sites and numbering corresponds to mouse TTP. OA, okadaic acid.

See also Figure S7.